Enediynes are a class of natural organic compounds that have been investigated for their potential as anti- cancer agents. Under appropriate reducing conditions, their unique structure predisposes them to rearrangement, resulting in the formation of a diradical intermediate capable of resulting in DNA strand scission. However, historically, the compounds had been deemed too cytotoxic because they undergo rapid activation at euthermic temperatures, and as a result, their clinical utility has been limited. Through manipulation of synthetic structures, the distance between alkyne termini, along with the electronic structure and redox status of the chromophoric region proximal to the reactive subunit are the critical factors that influence the activation temperatures of enediyne motifs. Since transition metals binding can modulate the geometric conformation and electronic structure of the organic enediyne ligand, metal ions are excellent cofactors for enediyne activation. Our laboratories constructed metallated enediyne motifs (metalloenediynes, ME) that display tightly-controlled thermal reactivity, and which may lead to increased future clinical utility. Specifically, we have developed and initiated the characterization of a suite of first-generation ME compounds that display a proclivity for activation after exposure to slightly supra-physiological temperatures. In this application, we shall tune the factors that influence Bergman cyclization in ME compounds, and evaluate their biological activities using in vitro and in vivo model systems. Our preliminary data indicate that unlike their natural product analogues, these novel MEs are not cytotoxic when administered to human normal and tumor cell lines at euthermic temperatures, but become highly cytotoxic when administered to cells at elevated temperatures. In addition, we have found that the compounds are not toxic when administered to mice, indicating that the compounds could be particularly useful as adjuvants to thermal therapy (hyperthermia or thermal ablation); they can be administered systemically with cytotoxicity confined to localized regions of heating. We shall determine the mechanisms by which heat enhances the cytotoxicity of MEs in melanoma and breast cancer cell lines, and utilize murine xenograft models to assess the potential clinical applicability of first-generation constructs already in hand. Through correlation of biological effects (e.g., cytotoxicity, DNA binding and damage, inhibition of DNA replication) with construct design, we also propose to develop new and optimized ME motifs, based on the Cisplatin design scaffold, that will show greater efficacy in killing tumor cells after brief exposures to fever-range or mild hyperthermic temperatures that are more easily achievable in the clinic compared to traditional hyperthermia treatments.